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Formononetin protects TBI rats against neurological lesions and the underlying mechanism
Journal of the Neurological Sciences 338 (2014) 112–117
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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Formononetin protects TBI rats against neurological lesions and the underlying mechanism Zhengzhao Li a, Xianhong Dong b, Jianfeng Zhang a, Guang Zeng a, Huimin Zhao a, Yun Liu e, Rubiao Qiu c, Linjian Mo d, Yu Ye a,⁎ a
Emergency Department, Western Hospital, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530007, PR China Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang 453003, PR China Guangxi Matemal and Child Health Hospital, Nanning, Guangxi Zhuang Autonomous Region 530003, PR China d Institute of Urology and Nephrology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China e Spine and Osteopathy Surgery Division, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China b c
a r t i c l e
i n f o
Article history: Received 1 November 2013 Received in revised form 7 December 2013 Accepted 17 December 2013 Available online 27 December 2013 Keywords: Formononetin Traumatic brain injury Rat Inflammation Oxidative stress Neuroprotection
a b s t r a c t Traumatic brain injury (TBI) is a major cause of disability or death worldwide, especially in the young. Thus, effective medication with few side effects needs to be developed. This work aimed to explore the potential benefits of formononetin (FN) on TBI rodent model and to discuss the regarding mechanism. These findings showed that FN effectively increased the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in brain tissue of TBI rats (P b 0.01), while it reduced intracephalic malonaldehyde (MDA), tumor necrosis factorα (TNF-α) and interleukin-6 (IL-6) concentrations (P b 0.01). Meanwhile, the hydrocephalus in theTBI rat was alleviated, and the injured nerve cell of the lesioned brain was reduced as showed in hematoxylin–eosin (HE) staining assay. In addition, the endogenous mRNA level of cyclooxygenase-2 (COX-2) in the brain of the TBI rat was significantly down-regulated (P b 0.01). Furthermore, the protein expression of nuclear factor E2-related factor 2 (Nrf2) was effectively up-regulated (P b 0.01). Taken together, we conclude that formononetin mediates the promising anti-TBI effects against neurocyte damage, which the underlying mechanisms are associated with inhibiting intracephalic inflammatory response and oxidative stress for neuroprotection. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Traumatic brain injury (TBI), also termed intracranial injury, results from the external pressure that traumatically occurs in the brain [1]. Brain trauma can induce secondary injury during the minutes and days. And TBI results in the abnormal changes of physical, emotional, cognitive, and behavior. More severely, the long-term conditions of moderate to severe TBI may appear in permanent disability or death [2]. The most common causes of TBI include traffic accidents, violence and other traumas [3]. Head injuries involved the direct contusion or indirect laceration is the leading cause of focal damages. If not being treated timely, the condition can usually deteriorate into diffuse injuries [4]. Physiologically, cascaded events of TBIrelated secondary injury are associated with the blood–brain barrier destruction, brain swelling, inflammatory mediator release, excessive free radical, excitotoxicity overload, and mitochondrial dysfunction [5,6]. It is very important to adopt promptly effective treatment once the TBI occurs, in which the wounded patient with moderate or severe injuries should be hospitalized in an intensive care unit to receive the
⁎ Corresponding author. Tel.: +86 771 3277068; fax: +86 771 3277161. E-mail address: [email protected] (Y. Ye). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.12.027
neurosurgical treatments [7]. Unfortunately, some existing clinical medications lead to the drug resistance into TBI patients. Meanwhile, there is accompanying with the unwanted side effects, such as decreasing consciousness [8]. Therefore, the potential effective substitutive needs to be exploited for managing post-traumatic symptoms, especially. Formononetin (FN) is a phytoestrogen extracted from plant-derived red clover, which it possesses the potent pharmacological activities, such as improving blood microcirculation, inducing apoptosis in cancer cells, and regulating antioxidant effects [9–11]. However, molecular mechanism of FN-attenuated neuronal lesions of TBI rats remains unknown. Thus, in the present study, we used a TBI rodent model to investigate the potential efficacy and mechanism of FN-mediated anti-TBI through analyzing cytokines related to inflammation and oxidative stress.
2. Materials and methods 2.1. Materials Formononetin (FN, purity N 98.0%; molecular formula as shown in Fig. 1A) was provided by Sigma Corporation of America. Other required materials were labeled below.
Z. Li et al. / Journal of the Neurological Sciences 338 (2014) 112–117
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Fig. 1. (A) The structure of formononetin (FN). (B) FN improved neurological conditions of TBI rat. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
2.2. Experimental animals and formononetin treatment
2.5. Evaluation of hydrocephalus condition
Male Wistar rats, mostly weighing 210 ± 5 g, were obtained from Guangxi Medical University, Laboratory Animal Center, China (License No. SYKG (Gui) 2003–0005). The TBI rat model was established as previously reported [12] with some modifications. In brief, the tested rats were anesthetized with the intraperitoneal injection of fresh 5% sodium pentobarbital solution (10 mL·kg− 1), then sterilely performing the experimental process: dissecting a 5 mm diameter incision in the skull (cerebral location: dextral coronal section at 1 mm, midline scalp at 2 mm). Subsequently, traumatic injury was conducted through striking using an impactor, with a 40 g hammer by freefall from 30 cm high to bump the endocranium (the bump-injured power was 0.05 kg·m− 1 s− 1), then sterilely suturing the scalp. The TBI rat model was identified successfully, as shown in limb convulsion, transitory apnea, and unconsciousness at post-injury. And then the experimental rats were randomly arranged into different groups: 10 healthy rats in sham-surgery control group (only the skull window being exposed); 10 TBI rats in model control group; 10 TBI rats in FN administrated groups (ip, 10 mg·kg − 1·d − 1 , 20 mg·kg− 1 ·d − 1 , for 5 days). All experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Guangxi Medical University.
The injured brain tissues of the TBI rat were harvested and measured the wet weight using an analytical balance, then recording the dry weight through being dewatered into the oven at 85 °C for 24 h to constant weight. Moisture content in the brain was calculated through wet–dry formula: Cerebral moisture content (%) = (wet weight − dry weight) / wet weight × 100%.
2.3. Assessment of neurological conditions Neurological function of the tested rats was investigated using the Neurological Severity Score according to Mahmood's method at post-TBI [13].
2.4. Brain tissue sampling preparation The rats were sacrificed using the intraperitoneal injection of overdose ethyl carbamate (200 mg·kg − 1). Intracephalic perfusion was conducted with 250 mL cold saline to remove the remanent blood. Brain tissues from the lesioned zone of each group were collected. Some samples were used for biochemical assays and histological inspection and others were employed for PCR and western blotting analyses, respectively.
2.6. Histopathological examination The paraffin sections were subjected to dewaxing and hydration, and then conducting hematoxylin and eosin stain (H&E stain) for 5 min. The slices were differentiated with 0.6% hydrochloric acid alcohol for 30 s, then counterstained in acidified eosin alcohol (pH 4.2) for 3 min, dehydrated and cleared, respectively. The pathological lesions zones of the brain were observed by a light microscope (Leica, Wetzlar, Germany). 2.7. Measurement of intracephalic regulatory enzymes Brain tissues of injured zone were homogenized with ice Tris–HCl (2 mmol/L containing 1 mmol/L EDTA, pH7.2), then centrifuging at 8000 ×g for 10 min at 4 °C. Aliquot supernatants were immediately utilized for the determination of GSH-Px, SOD, MDA, TNF-α and IL-6. All of these regulatory enzymes were measured according to the manufacturer's instructions (Boster, Wuhan, China). And the final units represented as nmol/mg protein or pg/mg protein, respectively. 2.8. Reverse transcription-polymerase chain reaction (RT-PCR) assay for COX-2 mRNA expression in brain tissues Frozen samples from injured zones of the brain were homogenized and total RNA was extracted with Trizol solution (Beyotime Institute of Biotechnology, Shanghai, China). RT-PCR was conducted using a commercial kit (Life Technologies, USA) according to manufacturer protocols. And internal β-actin was normalized as a control for the PCR reaction. Primer sequences (provided by Invitrogen Ltd.) were as follows: COX-2 sense: 5′-CCG CAG TAC AGA AAG TAT CAC-3′; antisense: 5′-ACA GCC CTT CAC GTT ATT G-3′ (401 bp); β-actin sense: 5′-GGG CGA ACA GGG TCA TCA TC-3′; antisense: 5′- ACC CTG GTC CTT AGT
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GGA GC-3′ (424 bp). Amplification of COX-2 mRNA was conducted through 30 cycles, including 1 min at 93 °C for denaturing, 30 s at 54 °C for annealing and 2 min at 70 °C for extension, while the annealing temperatures of COX-2 and β-actin were 49 °C and 54 °C, respectively. The ratio of COX-2/β-actin was evaluated by calculating the absorbance value of the targeting bands using the Quantity One 4.62 software (Bio-Rad, CA, USA). 2.9. Western blotting analysis for Nrf2 expression In brief, the cerebral tissues from lesioned zone were separated and homogenized with the RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) to extract intracellular protein following by centrifugation at 10,000 r/min for 10 min at 4 °C. Supernatant protein content was determined using a bicinchoninic acid assay protocols (Sigma-Aldrich, USA). Aliquot amounts of protein (50 μg) per lane were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to polyvinylidene difluoride (PVDF) membranes (Sigma-Aldrich, USA). After blocking, the primary antibody dilute solution (Santa Cruz, CA, USA; 1:2000) is incubated with the membrane under gentle agitation. And the membranes were then incubated with rabbit anti-Nrf2 monoclonal antibody (Santa Cruz, CA, USA; 1:4000), and mouse anti-rat β-actin antibody (Santa Cruz, CA, USA; 1:4000), respectively. Immunoreactive band was scanned and visualized using an enhanced chemiluminescence detection system (Life Technologies, USA) and X-ray film. Optical absorbance of band was quantified with the computer-assisted image analysis system (Bio-Rad, USA), and normalized to β-actin protein level. 2.10. Statistical analysis SPSS 13.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis in this study. Statistical differences between normal groups were analyzed using one-way analysis of variance (ANOVA) following by unpaired Student's t-test, and results were expressed as the means ± standard deviation (SD). The P b 0.05 was considered statistically significant. 3. Results
Fig. 2. FN decreased the cerebral moisture content of TBI rats. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
extracellular space between cells and cells was normally distributed. In contrast, TBI rats resulted in severe pathological alterations, such as cytoarchitecture destruction, vasocongestion, necrosis, inflammatory infiltration, and neuronal reduction. After the treatments of FN, the edema and necrosis in lesioned zones of the brain were significantly attenuated, and neural cell number was effectively increased when compared with the TBI control group (Fig. 3). 3.4. FN enhanced the antioxidant capability and reduced the inflammatory reaction in the lesioned brain of TBI rats To assess homoeostasis in FN-treated TBI rats, intracephalic levels of regulatory enzymes were determined, respectively. The results showed that the GSH-Px and SOD activities expressed in the lesioned brain zone of the TBI rat were obviously decreased, whereas the levels of MDA, TNF-α and IL-6 concentrations were elevated (P b 0.01, n = 10). Interestingly, FN-treated rats contributed to the increment of intracephalic GSH-Px and SOD activities and the reduction of MDA, TNF-α and IL-6 contents, respectively (P b 0.01, n = 10) (Fig. 4A–C).
3.1. FN ameliorated the neurological symptoms of TBI rat First, we investigated the neurological behaviors of FN-treated TBI rats. Compared with the sham control group, TBI rats resulted in significant neurological deficits, including the dystasia, trance and unconsciousness (P b 0.01, n = 10). Encouragingly, FN-treated TBI rats reversed these abnormal changes, as shown in improvement of mental conditions when compared with the TBI rat (P b 0.01, n = 10) (Fig. 1B). 3.2. FN reduced the cerebral moisture content of TBI rats In order to analyze TBI-induced cerebral hemorrhage in the experimental rats, intracephalic moisture content was evaluated. As shown in Fig. 2, the results indicated that hydrocephalus markedly occurred in TBI rats, which was higher than that in sham control group (P b 0.01, n = 10). Compared with the TBI rats, cerebral moisture content was gradually lowered following the FN treatments (P b 0.01, n = 10). 3.3. FN mitigated the pathological lesions in brain tissue of TBI rats Histological changes of rats from each group were observed using the hematoxylin–eosin (HE) staining assay. Nerve cells of brain tissue of sham control group were arranged uniformly, accompanied by the abundant cytoplasm and numerous neurocytes. In addition,
3.5. FN down-regulated the expression of COX-2 mRNA in the lesioned brain of TBI rats Next, we investigated the change of inflammation-related inducible enzyme in experimental rats. The findings showed that TBI rats appeared in elevated level of COX-2 mRNA in lesioned zone of the brain, which the expression was higher than that in the rats of sham control group (P b0.01, n = 10). Rather, the up-regulated COX-2 mRNA expression in TBI rats was inhibited by the FN treatment, as shown in reduction of COX-2 mRNA level in the lesioned brain (P b 0.01, n = 10) (Fig. 5). 3.6. FN increased the protein level of Nrf2 in the lesioned brain of TBI rats Further, antioxidant protein associated with the oxidative stress was determined (P b 0.01, n = 10). As a result, TBI rats showed the lowered Nrf2 protein expression in injured zone of the brain (P b 0.01, n = 10). Strikingly, FN-treated TBI rats contributed to the up-regulation of Nrf2 protein level in the lesioned brain, which the expression was more than that in TBI rats (P b 0.01, n = 10) (Fig. 6). 4. Discussion In clinic, patients suffered moderate to severe TBI generally receive routine pharmacological treatment after a neurosurgical operation
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Fig. 3. FN alleviated pathological injuries in brain tissue of lesioned TBI rats. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
[14]. The primary aim of medical prophylaxis is to prevent further neurotrauma from initial brain damage [15]. Pathologically, intracerebral hemorrhage is the endogenic destruction that can commonly deteriorate into diffuse injuries, which intracephalic bleeds itself is an intraaxial impairment originated from the penetration head trauma [16]. Thus, effectively managing intracerebral hemorrhage is an important measure in improving TBI conditions. In this study, the cerebral moisture content of TBI rats was markedly elevated that was more than that in sham control group, which indicated that hydrocephalus was accompanying with the traumatical brain hemorrhage. Beneficially, FNtreated TBI rats showed the decrease in hydrops in brain tissue at a dose-dependent manner, simultaneously ameliorating neurological symptoms as revealed in neurological evaluation score. Therefore, we preliminarily evidenced that FN exerted the cerebroprotective role against TBI. Inflammation occurred in central nervous system (CNS), usually termed neuroinflammation, is characterized by activated glial cell number, increased pro-inflammatory cytokine production, expanded blood– brain-barrier permeability, and eosinophilic granulocyte infiltration [17,18]. One key effector driven the neuroinflammation-linked initiation, a pro-inflammatory cytokine, refers to TNF-α, that is an abnormal increase in the injured brain [19]. Inflammatory signal is triggered through the activation of TNF-α/TNFR1 accessory protein complex, resulting in NF-κB-dependent transcription of pro-inflammatory cytokines, such as interleukins, chemokines, and interferons [20]. Under normal physiology conditions, cyclooxygenase-2 (simply called COX2) maintains lower level in tissues, while overexpression of COX-2
appears during the inflammatory reaction [21]. The COX-2 expression is significantly associated with various inflammatory subtypes, which in turn can stimulate the formation and release of regarding cytokines [22]. Therefore, pharmacological inhibition of intracellular COX-2 expression can beneficially perform the prevention and treatment of series of inflammation types. In the present study, our results showed that TBI rats resulted in elevation of TNF-α and IL-6 contents and upregulation of COX-2 mRNA level in impaired zone of brain, which these outcomes were consistent with the pronounced hydrocephalus in model rats. Thus, we speculate that intracerebral inflammatory response accelerates TBI deterioration. Following the FN treatments, pro-inflammatory cytokine concentrations of the injured brain were significantly reduced, accompanying with the corresponding decrease in COX-2 mRNA expression. These findings suggested that FN-mediated neuroprotection against the lesioned brain was involved in the mechanism as below. The intracephalic inflammatory mediators (such as TNF-α and IL-6) were effectively decreased through FN—inhibiting the COX-2 activities that occurred in astrocytes, neurones and microglia. We concluded that the modulation of inflammatory cascade in central nervous system (CNS) could be a therapeutic target for TBI. Scientifically, unregulated oxidative stress can produce excessive free radicals in body, gradually causing damage or death to the cells [23]. In turn, a substance with antioxidant activity conducts the termination of oxidative impairment via eliminating free radical intermediates or blocking other oxidative reactions [24]. Due to oxidative stress commonly occurring in various human diseases, the application of
Fig. 4. FN increased the antioxidant capacity (A–B) and decreased the inflammatory cytokines (C) in the lesioned brain of TBI rats. Results are presented as the mean ± SD (n = 10). a P b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
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Fig. 5. FN down-regulated the COX-2 mRNA expression in the lesioned brain of TBI rats. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, b P b 0.01 compared with TBI model group.
antioxidants in pharmacological field can benefit from treating the neurological disorders [25]. Glutathione peroxidase (GSH-Px) is an antioxidant enzyme, in which the main biochemical function is associated with protecting the cells against oxidative damage [26]. Superoxide dismutase (SOD) has multiple pharmacological activities. For example, SOD attenuates the reactive oxygen species (ROS) generation and oxidative stress development in CNS, thus alleviating neurodegenerative disorder [27]. Malondialdehyde (MDA) generation induced by lipotoxicity stress originates from the ROSmediated polyunsaturated lipid degradation [28–30]. Nuclear factor E2-related factor 2 (Nrf2), the antioxidant regulator, is a transcription factor that contributes to protecting against the cytotoxicity that is caused by oxidative stress [31]. When oxidative stress occurs, Nrf2 is activated, and then translocated into the nucleus where it combines to the DNA promoter and triggers the transcription of antioxidative genes and synthesis regarding proteins [32]. The main physiological functions of Nrf2 are involved in defending against series of pathologies, including atherosclerosis, chronic kidney injury, and neurodegeneration [33–35]. Hence, inhibition of intracephalic oxidative stress for maintaining the balance of oxidative defense system is an attractive therapeutic measure of anti-TBI. Our observations indicated that oxidative stress occurred in TBI rats, accompanied by neural lesions and neurocyte loss as shown in HE staining assay. Interestingly, these abnormal conditions were reversed following the FN treatments, accompanied by elevation of GSH-Px and SOD activities, Nrf2 protein and reduction of MDA content in impaired zone of brain. Taken
together, we speculated that TBI resulted in antioxidant capability insufficiency of brain, thereupon inducing inflammation and neural impairments. FN-mediated activation of Nrf2 pathway contributes to the induction of numerous cytoprotective proteins, in which these process perform series of biochemical cascading events for regulating antioxidative gene (such as GSH-Px, SOD) transcription to block the neuroinflammation and to exert the neuroprotection. In addition, Nrf2, as a drug target, could be identified for the treatment of TBI disease. 5. Conclusion This study demonstrates that FN treatment produces the promising finding for alleviation of cerebral trauma. A preliminary investigation of the underlying mechanism is involved in FN-mediated neuroprotection against TBI, which is through reducing inflammation and enhancing antioxidant capability in the lesioned brain. Overall, FN serves as the effective strategy of the prevention and treatment of anti-cerebral trauma. Declaration of interest None declared. Acknowledgments We thank Dr. Rubiao Qiu, Guangxi Matemal and Child Health Hospital for his help.
Fig. 6. FN up-regulated the Nrf2 protein level in the lesioned brain of TBI rats. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
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Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Formononetin protects TBI rats against neurological lesions and the underlying mechanism Zhengzhao Li a, Xianhong Dong b, Jianfeng Zhang a, Guang Zeng a, Huimin Zhao a, Yun Liu e, Rubiao Qiu c, Linjian Mo d, Yu Ye a,⁎ a
Emergency Department, Western Hospital, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530007, PR China Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang 453003, PR China Guangxi Matemal and Child Health Hospital, Nanning, Guangxi Zhuang Autonomous Region 530003, PR China d Institute of Urology and Nephrology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China e Spine and Osteopathy Surgery Division, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China b c
a r t i c l e
i n f o
Article history: Received 1 November 2013 Received in revised form 7 December 2013 Accepted 17 December 2013 Available online 27 December 2013 Keywords: Formononetin Traumatic brain injury Rat Inflammation Oxidative stress Neuroprotection
a b s t r a c t Traumatic brain injury (TBI) is a major cause of disability or death worldwide, especially in the young. Thus, effective medication with few side effects needs to be developed. This work aimed to explore the potential benefits of formononetin (FN) on TBI rodent model and to discuss the regarding mechanism. These findings showed that FN effectively increased the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in brain tissue of TBI rats (P b 0.01), while it reduced intracephalic malonaldehyde (MDA), tumor necrosis factorα (TNF-α) and interleukin-6 (IL-6) concentrations (P b 0.01). Meanwhile, the hydrocephalus in theTBI rat was alleviated, and the injured nerve cell of the lesioned brain was reduced as showed in hematoxylin–eosin (HE) staining assay. In addition, the endogenous mRNA level of cyclooxygenase-2 (COX-2) in the brain of the TBI rat was significantly down-regulated (P b 0.01). Furthermore, the protein expression of nuclear factor E2-related factor 2 (Nrf2) was effectively up-regulated (P b 0.01). Taken together, we conclude that formononetin mediates the promising anti-TBI effects against neurocyte damage, which the underlying mechanisms are associated with inhibiting intracephalic inflammatory response and oxidative stress for neuroprotection. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Traumatic brain injury (TBI), also termed intracranial injury, results from the external pressure that traumatically occurs in the brain [1]. Brain trauma can induce secondary injury during the minutes and days. And TBI results in the abnormal changes of physical, emotional, cognitive, and behavior. More severely, the long-term conditions of moderate to severe TBI may appear in permanent disability or death [2]. The most common causes of TBI include traffic accidents, violence and other traumas [3]. Head injuries involved the direct contusion or indirect laceration is the leading cause of focal damages. If not being treated timely, the condition can usually deteriorate into diffuse injuries [4]. Physiologically, cascaded events of TBIrelated secondary injury are associated with the blood–brain barrier destruction, brain swelling, inflammatory mediator release, excessive free radical, excitotoxicity overload, and mitochondrial dysfunction [5,6]. It is very important to adopt promptly effective treatment once the TBI occurs, in which the wounded patient with moderate or severe injuries should be hospitalized in an intensive care unit to receive the
⁎ Corresponding author. Tel.: +86 771 3277068; fax: +86 771 3277161. E-mail address: [email protected] (Y. Ye). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.12.027
neurosurgical treatments [7]. Unfortunately, some existing clinical medications lead to the drug resistance into TBI patients. Meanwhile, there is accompanying with the unwanted side effects, such as decreasing consciousness [8]. Therefore, the potential effective substitutive needs to be exploited for managing post-traumatic symptoms, especially. Formononetin (FN) is a phytoestrogen extracted from plant-derived red clover, which it possesses the potent pharmacological activities, such as improving blood microcirculation, inducing apoptosis in cancer cells, and regulating antioxidant effects [9–11]. However, molecular mechanism of FN-attenuated neuronal lesions of TBI rats remains unknown. Thus, in the present study, we used a TBI rodent model to investigate the potential efficacy and mechanism of FN-mediated anti-TBI through analyzing cytokines related to inflammation and oxidative stress.
2. Materials and methods 2.1. Materials Formononetin (FN, purity N 98.0%; molecular formula as shown in Fig. 1A) was provided by Sigma Corporation of America. Other required materials were labeled below.
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Fig. 1. (A) The structure of formononetin (FN). (B) FN improved neurological conditions of TBI rat. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
2.2. Experimental animals and formononetin treatment
2.5. Evaluation of hydrocephalus condition
Male Wistar rats, mostly weighing 210 ± 5 g, were obtained from Guangxi Medical University, Laboratory Animal Center, China (License No. SYKG (Gui) 2003–0005). The TBI rat model was established as previously reported [12] with some modifications. In brief, the tested rats were anesthetized with the intraperitoneal injection of fresh 5% sodium pentobarbital solution (10 mL·kg− 1), then sterilely performing the experimental process: dissecting a 5 mm diameter incision in the skull (cerebral location: dextral coronal section at 1 mm, midline scalp at 2 mm). Subsequently, traumatic injury was conducted through striking using an impactor, with a 40 g hammer by freefall from 30 cm high to bump the endocranium (the bump-injured power was 0.05 kg·m− 1 s− 1), then sterilely suturing the scalp. The TBI rat model was identified successfully, as shown in limb convulsion, transitory apnea, and unconsciousness at post-injury. And then the experimental rats were randomly arranged into different groups: 10 healthy rats in sham-surgery control group (only the skull window being exposed); 10 TBI rats in model control group; 10 TBI rats in FN administrated groups (ip, 10 mg·kg − 1·d − 1 , 20 mg·kg− 1 ·d − 1 , for 5 days). All experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Guangxi Medical University.
The injured brain tissues of the TBI rat were harvested and measured the wet weight using an analytical balance, then recording the dry weight through being dewatered into the oven at 85 °C for 24 h to constant weight. Moisture content in the brain was calculated through wet–dry formula: Cerebral moisture content (%) = (wet weight − dry weight) / wet weight × 100%.
2.3. Assessment of neurological conditions Neurological function of the tested rats was investigated using the Neurological Severity Score according to Mahmood's method at post-TBI [13].
2.4. Brain tissue sampling preparation The rats were sacrificed using the intraperitoneal injection of overdose ethyl carbamate (200 mg·kg − 1). Intracephalic perfusion was conducted with 250 mL cold saline to remove the remanent blood. Brain tissues from the lesioned zone of each group were collected. Some samples were used for biochemical assays and histological inspection and others were employed for PCR and western blotting analyses, respectively.
2.6. Histopathological examination The paraffin sections were subjected to dewaxing and hydration, and then conducting hematoxylin and eosin stain (H&E stain) for 5 min. The slices were differentiated with 0.6% hydrochloric acid alcohol for 30 s, then counterstained in acidified eosin alcohol (pH 4.2) for 3 min, dehydrated and cleared, respectively. The pathological lesions zones of the brain were observed by a light microscope (Leica, Wetzlar, Germany). 2.7. Measurement of intracephalic regulatory enzymes Brain tissues of injured zone were homogenized with ice Tris–HCl (2 mmol/L containing 1 mmol/L EDTA, pH7.2), then centrifuging at 8000 ×g for 10 min at 4 °C. Aliquot supernatants were immediately utilized for the determination of GSH-Px, SOD, MDA, TNF-α and IL-6. All of these regulatory enzymes were measured according to the manufacturer's instructions (Boster, Wuhan, China). And the final units represented as nmol/mg protein or pg/mg protein, respectively. 2.8. Reverse transcription-polymerase chain reaction (RT-PCR) assay for COX-2 mRNA expression in brain tissues Frozen samples from injured zones of the brain were homogenized and total RNA was extracted with Trizol solution (Beyotime Institute of Biotechnology, Shanghai, China). RT-PCR was conducted using a commercial kit (Life Technologies, USA) according to manufacturer protocols. And internal β-actin was normalized as a control for the PCR reaction. Primer sequences (provided by Invitrogen Ltd.) were as follows: COX-2 sense: 5′-CCG CAG TAC AGA AAG TAT CAC-3′; antisense: 5′-ACA GCC CTT CAC GTT ATT G-3′ (401 bp); β-actin sense: 5′-GGG CGA ACA GGG TCA TCA TC-3′; antisense: 5′- ACC CTG GTC CTT AGT
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GGA GC-3′ (424 bp). Amplification of COX-2 mRNA was conducted through 30 cycles, including 1 min at 93 °C for denaturing, 30 s at 54 °C for annealing and 2 min at 70 °C for extension, while the annealing temperatures of COX-2 and β-actin were 49 °C and 54 °C, respectively. The ratio of COX-2/β-actin was evaluated by calculating the absorbance value of the targeting bands using the Quantity One 4.62 software (Bio-Rad, CA, USA). 2.9. Western blotting analysis for Nrf2 expression In brief, the cerebral tissues from lesioned zone were separated and homogenized with the RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) to extract intracellular protein following by centrifugation at 10,000 r/min for 10 min at 4 °C. Supernatant protein content was determined using a bicinchoninic acid assay protocols (Sigma-Aldrich, USA). Aliquot amounts of protein (50 μg) per lane were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to polyvinylidene difluoride (PVDF) membranes (Sigma-Aldrich, USA). After blocking, the primary antibody dilute solution (Santa Cruz, CA, USA; 1:2000) is incubated with the membrane under gentle agitation. And the membranes were then incubated with rabbit anti-Nrf2 monoclonal antibody (Santa Cruz, CA, USA; 1:4000), and mouse anti-rat β-actin antibody (Santa Cruz, CA, USA; 1:4000), respectively. Immunoreactive band was scanned and visualized using an enhanced chemiluminescence detection system (Life Technologies, USA) and X-ray film. Optical absorbance of band was quantified with the computer-assisted image analysis system (Bio-Rad, USA), and normalized to β-actin protein level. 2.10. Statistical analysis SPSS 13.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis in this study. Statistical differences between normal groups were analyzed using one-way analysis of variance (ANOVA) following by unpaired Student's t-test, and results were expressed as the means ± standard deviation (SD). The P b 0.05 was considered statistically significant. 3. Results
Fig. 2. FN decreased the cerebral moisture content of TBI rats. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
extracellular space between cells and cells was normally distributed. In contrast, TBI rats resulted in severe pathological alterations, such as cytoarchitecture destruction, vasocongestion, necrosis, inflammatory infiltration, and neuronal reduction. After the treatments of FN, the edema and necrosis in lesioned zones of the brain were significantly attenuated, and neural cell number was effectively increased when compared with the TBI control group (Fig. 3). 3.4. FN enhanced the antioxidant capability and reduced the inflammatory reaction in the lesioned brain of TBI rats To assess homoeostasis in FN-treated TBI rats, intracephalic levels of regulatory enzymes were determined, respectively. The results showed that the GSH-Px and SOD activities expressed in the lesioned brain zone of the TBI rat were obviously decreased, whereas the levels of MDA, TNF-α and IL-6 concentrations were elevated (P b 0.01, n = 10). Interestingly, FN-treated rats contributed to the increment of intracephalic GSH-Px and SOD activities and the reduction of MDA, TNF-α and IL-6 contents, respectively (P b 0.01, n = 10) (Fig. 4A–C).
3.1. FN ameliorated the neurological symptoms of TBI rat First, we investigated the neurological behaviors of FN-treated TBI rats. Compared with the sham control group, TBI rats resulted in significant neurological deficits, including the dystasia, trance and unconsciousness (P b 0.01, n = 10). Encouragingly, FN-treated TBI rats reversed these abnormal changes, as shown in improvement of mental conditions when compared with the TBI rat (P b 0.01, n = 10) (Fig. 1B). 3.2. FN reduced the cerebral moisture content of TBI rats In order to analyze TBI-induced cerebral hemorrhage in the experimental rats, intracephalic moisture content was evaluated. As shown in Fig. 2, the results indicated that hydrocephalus markedly occurred in TBI rats, which was higher than that in sham control group (P b 0.01, n = 10). Compared with the TBI rats, cerebral moisture content was gradually lowered following the FN treatments (P b 0.01, n = 10). 3.3. FN mitigated the pathological lesions in brain tissue of TBI rats Histological changes of rats from each group were observed using the hematoxylin–eosin (HE) staining assay. Nerve cells of brain tissue of sham control group were arranged uniformly, accompanied by the abundant cytoplasm and numerous neurocytes. In addition,
3.5. FN down-regulated the expression of COX-2 mRNA in the lesioned brain of TBI rats Next, we investigated the change of inflammation-related inducible enzyme in experimental rats. The findings showed that TBI rats appeared in elevated level of COX-2 mRNA in lesioned zone of the brain, which the expression was higher than that in the rats of sham control group (P b0.01, n = 10). Rather, the up-regulated COX-2 mRNA expression in TBI rats was inhibited by the FN treatment, as shown in reduction of COX-2 mRNA level in the lesioned brain (P b 0.01, n = 10) (Fig. 5). 3.6. FN increased the protein level of Nrf2 in the lesioned brain of TBI rats Further, antioxidant protein associated with the oxidative stress was determined (P b 0.01, n = 10). As a result, TBI rats showed the lowered Nrf2 protein expression in injured zone of the brain (P b 0.01, n = 10). Strikingly, FN-treated TBI rats contributed to the up-regulation of Nrf2 protein level in the lesioned brain, which the expression was more than that in TBI rats (P b 0.01, n = 10) (Fig. 6). 4. Discussion In clinic, patients suffered moderate to severe TBI generally receive routine pharmacological treatment after a neurosurgical operation
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Fig. 3. FN alleviated pathological injuries in brain tissue of lesioned TBI rats. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
[14]. The primary aim of medical prophylaxis is to prevent further neurotrauma from initial brain damage [15]. Pathologically, intracerebral hemorrhage is the endogenic destruction that can commonly deteriorate into diffuse injuries, which intracephalic bleeds itself is an intraaxial impairment originated from the penetration head trauma [16]. Thus, effectively managing intracerebral hemorrhage is an important measure in improving TBI conditions. In this study, the cerebral moisture content of TBI rats was markedly elevated that was more than that in sham control group, which indicated that hydrocephalus was accompanying with the traumatical brain hemorrhage. Beneficially, FNtreated TBI rats showed the decrease in hydrops in brain tissue at a dose-dependent manner, simultaneously ameliorating neurological symptoms as revealed in neurological evaluation score. Therefore, we preliminarily evidenced that FN exerted the cerebroprotective role against TBI. Inflammation occurred in central nervous system (CNS), usually termed neuroinflammation, is characterized by activated glial cell number, increased pro-inflammatory cytokine production, expanded blood– brain-barrier permeability, and eosinophilic granulocyte infiltration [17,18]. One key effector driven the neuroinflammation-linked initiation, a pro-inflammatory cytokine, refers to TNF-α, that is an abnormal increase in the injured brain [19]. Inflammatory signal is triggered through the activation of TNF-α/TNFR1 accessory protein complex, resulting in NF-κB-dependent transcription of pro-inflammatory cytokines, such as interleukins, chemokines, and interferons [20]. Under normal physiology conditions, cyclooxygenase-2 (simply called COX2) maintains lower level in tissues, while overexpression of COX-2
appears during the inflammatory reaction [21]. The COX-2 expression is significantly associated with various inflammatory subtypes, which in turn can stimulate the formation and release of regarding cytokines [22]. Therefore, pharmacological inhibition of intracellular COX-2 expression can beneficially perform the prevention and treatment of series of inflammation types. In the present study, our results showed that TBI rats resulted in elevation of TNF-α and IL-6 contents and upregulation of COX-2 mRNA level in impaired zone of brain, which these outcomes were consistent with the pronounced hydrocephalus in model rats. Thus, we speculate that intracerebral inflammatory response accelerates TBI deterioration. Following the FN treatments, pro-inflammatory cytokine concentrations of the injured brain were significantly reduced, accompanying with the corresponding decrease in COX-2 mRNA expression. These findings suggested that FN-mediated neuroprotection against the lesioned brain was involved in the mechanism as below. The intracephalic inflammatory mediators (such as TNF-α and IL-6) were effectively decreased through FN—inhibiting the COX-2 activities that occurred in astrocytes, neurones and microglia. We concluded that the modulation of inflammatory cascade in central nervous system (CNS) could be a therapeutic target for TBI. Scientifically, unregulated oxidative stress can produce excessive free radicals in body, gradually causing damage or death to the cells [23]. In turn, a substance with antioxidant activity conducts the termination of oxidative impairment via eliminating free radical intermediates or blocking other oxidative reactions [24]. Due to oxidative stress commonly occurring in various human diseases, the application of
Fig. 4. FN increased the antioxidant capacity (A–B) and decreased the inflammatory cytokines (C) in the lesioned brain of TBI rats. Results are presented as the mean ± SD (n = 10). a P b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
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Fig. 5. FN down-regulated the COX-2 mRNA expression in the lesioned brain of TBI rats. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, b P b 0.01 compared with TBI model group.
antioxidants in pharmacological field can benefit from treating the neurological disorders [25]. Glutathione peroxidase (GSH-Px) is an antioxidant enzyme, in which the main biochemical function is associated with protecting the cells against oxidative damage [26]. Superoxide dismutase (SOD) has multiple pharmacological activities. For example, SOD attenuates the reactive oxygen species (ROS) generation and oxidative stress development in CNS, thus alleviating neurodegenerative disorder [27]. Malondialdehyde (MDA) generation induced by lipotoxicity stress originates from the ROSmediated polyunsaturated lipid degradation [28–30]. Nuclear factor E2-related factor 2 (Nrf2), the antioxidant regulator, is a transcription factor that contributes to protecting against the cytotoxicity that is caused by oxidative stress [31]. When oxidative stress occurs, Nrf2 is activated, and then translocated into the nucleus where it combines to the DNA promoter and triggers the transcription of antioxidative genes and synthesis regarding proteins [32]. The main physiological functions of Nrf2 are involved in defending against series of pathologies, including atherosclerosis, chronic kidney injury, and neurodegeneration [33–35]. Hence, inhibition of intracephalic oxidative stress for maintaining the balance of oxidative defense system is an attractive therapeutic measure of anti-TBI. Our observations indicated that oxidative stress occurred in TBI rats, accompanied by neural lesions and neurocyte loss as shown in HE staining assay. Interestingly, these abnormal conditions were reversed following the FN treatments, accompanied by elevation of GSH-Px and SOD activities, Nrf2 protein and reduction of MDA content in impaired zone of brain. Taken
together, we speculated that TBI resulted in antioxidant capability insufficiency of brain, thereupon inducing inflammation and neural impairments. FN-mediated activation of Nrf2 pathway contributes to the induction of numerous cytoprotective proteins, in which these process perform series of biochemical cascading events for regulating antioxidative gene (such as GSH-Px, SOD) transcription to block the neuroinflammation and to exert the neuroprotection. In addition, Nrf2, as a drug target, could be identified for the treatment of TBI disease. 5. Conclusion This study demonstrates that FN treatment produces the promising finding for alleviation of cerebral trauma. A preliminary investigation of the underlying mechanism is involved in FN-mediated neuroprotection against TBI, which is through reducing inflammation and enhancing antioxidant capability in the lesioned brain. Overall, FN serves as the effective strategy of the prevention and treatment of anti-cerebral trauma. Declaration of interest None declared. Acknowledgments We thank Dr. Rubiao Qiu, Guangxi Matemal and Child Health Hospital for his help.
Fig. 6. FN up-regulated the Nrf2 protein level in the lesioned brain of TBI rats. Results are presented as the mean ± SD (n = 10). aP b 0.01 compared with sham control group, bP b 0.01 compared with TBI model group.
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